Antisense oligonucleotides inhibiting human bcl-2 gene expression

ABSTRACT

The present invention provides novel antisense oligodeoxynucleotides which are useful in inhibiting lymphoma or leukemia cells. The subject oligodeoxynucleotides are complementary to a strategic site in the mRNA sense strand to the human bcl-2 gene. Such oligodeoxynucleotide are provided either in their native state or as a derivative such as the phosphorothioate. In the preferred embodiments, these antisense oligodeoxynucleotide straddle the predicted translation-initiation site of the mRNA coding strand, the splice donor region, the splice acceptor region of the mRNA coding strand, or the 5&#39;-cap region for the human bcl-2 gene.

REFERENCE TO GOVERNMENT GRANTS

The research in this patent application was supported in part byNational Institutes of Health grant CA 26380. The United Statesgovernment has certain rights in the invention.

This application is a continuation of Ser. No. 07/840,716, filed Feb.21, 1992, abandoned, which is a continuation-in-part of Ser. No.07/288,692, filed Dec. 22, 1988 abandoned.

FIELD OF THE INVENTION

The present invention relates to the field of treatments for cancer andmore particularly to the field of antisense oligonucleotide treatmentsfor cancer.

BACKGROUND OF THE INVENTION

Current approaches to cancer treatment suffer from a lack ofspecificity. The majority of drugs that have been developed are naturalproducts or derivatives that either block enzyme pathways or randomlyinteract with DNA. Due to low therapeutic indices, most cancer treatmentdrugs are accompanied by serious dose-limiting toxicities. Theadministration of drugs to treat cancer kills not only cancer cells butalso normal non-cancerous cells. Because of these deleterious effects,treatments that are more specific for cancerous cells are needed.

It has been found that a class of genes, the oncogenes, plays a largerole in the transformation and maintenance of the cancerous state andthat turning off these genes, or otherwise inhibiting their effects, canreturn a cell to a normal phenotype. The role of oncogenes in theetiology of many human cancers has been reviewed in Bishop, "CellularOncogenes and Retroviruses", Science, 235:305-311 (1987).

Recently, a human gene termed bcl-2 (B cell lymphoma/leukemia-2) hasbeen implicated in the etiology of some common lymphoid tumors, Croce etal., "Molecular Basis Of Human B and T Cell Neoplasia", in: Advance inViral Oncology, 7:35-51, G. Klein (ed.), New York: Raven Press, 1987.High levels of expression of the human bcl-2 gene have been found in alllymphomas with t (14; 18) chromosomal translocations including mostfollicular B cell lymphomas and many large cell non-Hodgkin's lymphomas.High levels of expression of the bcl-2 gene have also been found incertain leukemias that do not have a t (14; 18) chromosomaltranslocation, including most acute lymphocytic leukemias of the pre-Bcell type.

Antisense oligodeoxynucleotides are one example of a specifictherapeutic tool with the potential for ablating oncogene function.These short (usually about 30 bases) single-stranded synthetic DNAs havea complementary base sequence to the target mRNA and form a hybridduplex by hydrogen bonded base pairing. This hybridization can beexpected to prevent expression of the target mRNA code into its proteinproduct and thus preclude subsequent effects of the protein product.Because the mRNA sequence expressed by the gene is termed the sensesequence, the complementary sequence is termed the antisense sequence.Inhibition of mRNA would be more efficient than inhibition of anenzyme's active site, since one mRNA molecule gives rise to multipleprotein copies.

Synthetic oligodeoxynucleotides complementary to (antisense) mRNA of thec-myc oncogene have been used to specifically inhibit production ofc-myc protein, thus arresting the growth of human leukemic cells invitro, Holt et al., Mol. Cell Biol. 8:963-973 (1988), and Wickstrom etal., Proc. Natl. Acad. Scio USA, 85:1028-1-32 (1988).Oligodeoxynucleotides have also been employed as specific inhibitors ofretroviruses, including the human immunodeficiency virus (HIV-I),Zamecnik and Stephenson, Proc. Natl. Acad. Sci. USA, 75:280-284 (1978)and Zamecnik et al., Proc. Natl. Acad. Sci. USA, 83:4143-4146 (1986).

SUMMARY OF THE INVENTION

The invention provides oligodeoxynucleotides and methods for inhibitinggrowth of lymphoma or leukemia cells, that are types of lymphocytes. Anantisense oligodeoxynucleotide complementary to at least an effectiveportion of the mRNA sense strand to the human bcl-2 gene is provided andcells are then contacted with the antisense oligodeoxynucleotide in aconcentration sufficient to inhibit proliferation of the cells. Themethods of the invention are suitable for inhibiting proliferation oflymphoma/leukemia cells that express the human bcl-2 gene and have a t(14; 18) chromosomal translocation as well as those that express thebcl-2 gene but do not have a t (14; 18) chromosomal translocation.

In accordance with preferred embodiments, the oligodeoxynucleotide issubstantially complementary to a strategic site in the mRNA sensestrand. A preferred strategic site is the translation-initiation site ofthe mRNA coding strand. Alternative strategic sites include coding sitesfor splicing, transport or degradation. The subject antisenseoligodeoxynucleotide either in its "native", unmodified form or as aderivative, is brought into contact with the target lymphoma or leukemiacells. For in vivo therapeutic use, a derivative of the "native"oligodeoxynucleotide, such as the phosphorothioate form is preferredsince it is believed that these forms are more resistant to degradation,notwithstanding the fact that response times to some analogues, such asthe phosphorothioate analogs, has been found to be somewhat slower thanto the "native" form of the oligodeoxynucleotide.

A preferred antisense oligodeoxynucleotide, denominated herein the TI-AS(translation initiation antisense) nucleotide straddles thetranslation-initiation site of the mRNA coding strand of the human bcl-2gene and is complementary to this region. More preferably, thisnucleotide comprises a TAC portion which is complementary to the ATGinitiation sequence of the coding strand for the bcl-2 gene, andpreferably further comprises flanking portions of two to about onehundred bases, more preferably from about five to about twenty bases,which are complementary to portions of the bcl-2 gene coding strandflanking said initiation sequence. The TI-AS nucleotide has been foundeffective at inhibiting the growth of the target cells both in thepresence and absence of serum.

Alternatively, the antisense oligodeoxynucleotide comprises an antisensenucleotide complementary to at least an effective portion of the splicedonor site of the mRNA coding strand for the human bcl-2 gene. Moreparticularly, this nucleotide comprises a CA portion which iscomplementary to the GT splice donor of the bcl-2, and again comprisesflanking portions of two to about one hundred bases, preferably fromabout five to about twenty bases, which are complementary to portions ofthe bcl-2 gene coding strand flanking said splice donor.

In yet another embodiment, the antisense nucleotide is complementary toat least an effective portion of the splice acceptor region of the mRNAcoding strand for the human bcl-2 gene. This nucleotide comprises atleast a TC portion which is complementary to the AG splice acceptor ofthe bcl-2 gene, and again comprises flanking portions of two to aboutone hundred, preferably from about five to about twenty bases which arecomplementary to portions of the bcl-2 gene coding strand flanking saidacceptor. The subject oligodeoxynucleotide may also be selected tooverlap the coding site for the 26 kDa protein, bcl-2-alpha or for the22 kDa protein, bcl-2-beta, protein products of the bcl-2 gene.Preferably the oligodeoxynucleotide is selected to minimize homologywith antisense nucleotides for mRNA coding strands for other genesequences.

Accordingly, a primary object of the present invention is the provisionof novel antisense oligodeoxynucleotides which are useful in inhibitingthe growth of leukemia and/or lymphoma cells. A further object of thepresent invention is the provision of methods for inhibiting the growthof leukemia and/or lymphoma cells using said oligodeoxynucleotides.These and other objects of the present invention will become apparentfrom the following, more detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows graphs of the effects of varying concentrations ofoligodeoxynucleotides on inhibition of cell proliferation.

FIG. 2 shows graphs of the concentration dependence of inhibition ofcell proliferation by antisense normal and phosphorothioateoligodeoxynucleotides. Oligodeoxynucleotide additions to culturesincluded TI-AS phosphorothioate (o and ; two separate experiments),TI-S phosphorothioate (Δ), TI-AS normal (¤), and TI-S normal (Δ).

FIG. 3 shows the results of gel electrophoresis of six antisenseoligonucleotides targeted against the translation initiation site ofbcl-2 mRNA.

FIG. 4 shows the degree of DNA fragmentation resulting fromoligonucleotide treatment of RS11846 cells. FIG. 4 (a) shows the effectof oligonucleotides targeted against the translation initiation site.FIG. 4 (b) shows the effect of oligonucleotides directed against the5'-cap region of bcl-2 mRNA.

FIG. 5 is a graph showing the concentration-dependence of inhibition byan antisense oligonucleotide targeted against the translation initiationsite of bcl-2 mRNA.

FIGS. 6 (a) and (b) are graphs showing the results of immunofluorescenceanalysis of bcl-2 protein levels in oligonucleotide-treated cells.

FIGS. 7 (a)-(d) are FACS profiles for 697 cells before and aftertreatment with bcl-2 antisense oligonucleotides.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Oligodeoxynucleotides are selected for use in the invention according totheir ability to inhibit cell proliferation. Uncontrolled cellproliferation is a marker for a cancerous or abnormal cell type. Normal,non-cancerous cells divide regularly, at a frequency characteristic forthe particular type of cell. When a cell has been transformed into acancerous state, the cell divides and proliferates uncontrollably.Inhibition of proliferation modulates the uncontrolled division of thecell. Containment of cell division often correlates with a return to anon-cancerous state.

Oligodeoxynucleotides suitable for use in the invention are preferablytwo to two hundred nucleotide bases long; more preferably ten to fortybases long; most preferably twenty bases long. The oligodeoxynucleotidesare preferably selected from those oligodeoxynucleotides complementaryto strategic sites along the mRNA of bcl-2, such as the translationinitiation site, donor and acceptor splicing sites, or sites fortransportation or degradation. Blocking translation at such strategicsites prevents formation of a functional bcl-2 gene product. It shouldbe appreciated, however, that any combination or subcombination ofnucleotides complementary or substantially complementary to the bcl-2mRNA that inhibits cell proliferation is suitable for use in theinvention. For example, oligodeoxynucleotides complementary tonon-contiguous stretches of the bcl-2mRNA may inhibit cell proliferationand would thus be suitable for use in the invention. It should also beappreciated that oligodeoxynucleotides suitable for use in the inventionmay also include nucleotides flanking those complementary orsubstantially complementary to the strategic or other sites along thebcl-2 mRNA. The flanking portions are preferably from two to about onehundred bases, more preferably from about five to about twenty bases inlength. It is also preferable that the oligodeoxynucleotide becomplementary to a portion of the mRNA that is not commonly found inmRNA of other genes to minimize homology of antisense nucleotides formRNA coding strands from other genes.

Preferred antisense, or complementary, oligodeoxynucleotides are listedin Table 1.

                                      TABLE 1                                     __________________________________________________________________________    bcl-2 Oligodeoxynucleotides                                                   __________________________________________________________________________    transition initiation                                                         antisense (TI-AS)                                                                      3' . . . CCCTTCCTACCGCGTGCGAC . . . 5'  (SEQ ID NO: 1)               bcl-2    5' . . . CTTTTCCTCTGGGAAGGATGGCGCACGCTGGGAGA . . . 3'  (SEQ ID                NO: 2)                                                               splice donor                                                                  antisense (SD-AS)                                                                      3' . . . CCTCCGACCCATCCACGTAG . . . 5'  (SEQ ID NO: 3)               bcl-2    5' . . . ACGGGGTAC . . . GGAGGCTGGGTAGGTGCATCTGGT . . . 3'  (SEQ              ID NO: 4)                                                            splice acceptor                                                               antisense (SA-AS)                                                                      3' . . . GTTGACGTCCTACGGAAACA . . . 5'  (SEQ ID NO: 5)               bcl-2    5' . . . CCCCCAACTGCAGGATGCCTTTGTGGAACTGTACGG . . . 3'  (SEQ ID               NO: 6)                                                               __________________________________________________________________________

It will be appreciated by those skilled in the art to which thisinvention pertains, that oligodeoxynucleotides having a greater orlesser number of substituent nucleotides, or that extend further alongthe bcl-2 mRNA in either the 3' or 5' direction than the preferredembodiments, but which also inhibit cell proliferation are also withinthe scope of the invention.

It is preferable to use derivatives of antisense oligodeoxynucleotidesin the performance of the invention rather than "native" or unmodifiedoligodeoxynucleotides. "Native" oligodeoxynucleotides can beconveniently synthesized with a DNA synthesizer using standardphosphoramidite chemistry. Suitable derivatives, and methods forpreparing the derivatives, include phosphorothioate, Stein et al., Nucl.Acids Res., 16:3209-3221 (1988); methylphosphonate, Blake et al.,Biochemistry 24:6132-6138 (1985) and alpha-deoxynucleotides, Morvan etal., Nucl. Acids Res., 14:5019-5032 (1986) and covalently-linkedderivatives such as acridine, Asseline et al., Proc. Natl Acad. Sci. USA81:3297-3301 (1984); alkylated (e.g., N-2-chloroethylamine), Knorre etal., Biochemie 67:785-789 (1985) and Vlassov et al., Nucl. Acids Res.14:4065-4076 (1986); phenazine, Knorre et al., supra, and Vlassov etal., supra; 5-methyl-N⁴ -N⁴ -ethano-cytosine, Webb et al., Nucl. AcidsRes. 14:7661-7674 (1986); Fe-ethylenediamine tetraacetic acid (EDTA) andanalogues, Boutorin et al., FEBS Letters 172:43-46 (1984);5-glycylamido-1, 10-O-phenanthroline, Chi-Hong et al., Proc. Natl. Acad.Sci. USA 83:7147-7151 (1986); and diethylenetriamine-pentaacetic acid(DTPA) derivatives, Chu et al., Proc. Natl. Acad. Sci. USA 82:963-967(1985). All of the above publications are hereby specificallyincorporated by reference as if fully set forth herein.

The oligodeoxynucleotides may be administered to patients by anyeffective route, including intravenous, intramuscular, intrathecal,intranasal, intraperitoneal, subcutaneous injection, in situ injectionand oral administration. The oligodeoxynucleotides may be mixed with anamount of a physiologically acceptable carrier or diluent, such as asaline solution or other suitable liquid. The oligodeoxynucleotides mayalso be combined with liposomes or other carriers.

The oligodeoxynucleotides may also be useful for ex vivo bone marrowpurging. Normally, the amounts of conventional chemotherapeutic drugsand irradiation that a patient can receive are limited by toxicity tothe marrow, i.e., anemia (fatigue, heart failure), thrombocytopenia(bleeding), neutropenia (infection). Thus, in order to deliversufficient concentrations of drugs and irradiation to totally eradicatethe tumor, the physician would simultaneously destroy the patient'snormal bone marrow cells leading to patient demise. Alternatively, largeamounts of bone marrow can be surgically extracted from the patient andstored in vitro, while the patient receives aggressive conventionaltreatment. The patient can then be rescued by reinfusion of their ownbone marrow cells, but only if that marrow has been "purged" of residualmalignant cells. Antisense reagents employing the oligodeoxynucleotidesof the invention could be used to remove residual malignant cells fromthe bone marrow.

The oligodeoxynucleotides are administered in amounts effective toinhibit leukemia/lymphoma cell proliferation. The actual amount of anyparticular oligodeoxynucleotide administered will depend on factors suchas the type of lymphoma/leukemia, the toxicity of theoligodeoxynucleotide to other cells of the body, its rate of uptake byleukemia/lymphoma cells, and the weight and age of the individual towhom the oligodeoxynucleotide is administered. Because of inhibitorspresent in human serum that may interfere with the action of theoligodeoxynucleotides, an effective amount of the oligodeoxynucleotidesfor each individual may vary. An effective dosage for the patient can beascertained by conventional methods such as incrementally increasing thedosage of the oligodeoxynucleotide from an amount ineffective to inhibitcell proliferation to an effective amount. It is expected thatconcentrations presented to leukemia/lymphoma cells in the range ofabout 10 to about 400 micromolar, more preferably in the range of fromabout 20 micromolar to about 250 micromolar, will be effective toinhibit cell proliferation.

The oligodeoxynucleotides are administered to the patient for at least atime sufficient to inhibit proliferation of the lymphoma/leukemia cells.The oligodeoxynucleotides are preferably administered to patients at afrequency sufficient to maintain the level of oligodeoxynucleotides atan effective level in or around the lymphoma/leukemia cells. To maintainan effective level, it may be necessary to administer theoligodeoxynucleotides several times a day, daily or at less frequentintervals. Oligodeoxynucleotides are administered untillymphoma-leukemia cells can no longer be detected, or have been reducedin number such that further treatment provides no significant reductionin number, or the cells have been reduced to a number manageable bysurgery or other treatments. The length of time that theoligodeoxynucleotides are administered will depend on factors such asthe rate of uptake of the particular oligodeoxynucleotide bylymphoma/leukemia cells and time needed for the cells to respond to theoligodeoxynucleotide. In vitro, maximal inhibition of leukemic cellproliferation by "native", unmodified antisense oligodeoxynucleotidesoccurred two days after initiation of cultures, whereas phosphorothioateoligodeoxynucleotides required 4 to 7 days to achieve maximalinhibition. In vivo, the time necessary for maximal inhibition of cellproliferation may be shorter or longer.

The oligodeoxynucleotides of the invention may be administered topatients as a combination of two or more different antisenseoligodeoxynucleotide sequences or as a single type of sequence. Forinstance, TI-AS and SD-AS could be administered to a patient or TI-ASalone.

It is also believed that the oligodeoxynucleotides of the invention maybe useful in the treatment of autoimmune diseases. Autoimmune diseasesare those diseases in which the body's immune system has malfunctionedin some way. Administration of the oligodeoxynucleotides of theinvention to a person having an autoimmune disease should inhibitproliferation of lymphocytes which would in turn reduce the symptoms ofthe autoimmune disease. For use in treating autoimmune diseases, theoligodeoxynucleotides would be administered as described herein.

EXPERIMENTAL Preparation of Oligodeoxynucleotides

Normal and phosphorothioate oligodeoxynucleotides were synthesized usingan Applied Biosystems 380B DNA synthesizer, and purified by HPLCreverse-phase chromatography (PRP-1 column) as described in Stein etal., Nucl. Acids Res., 16:3209-3221 (1988) which is specificallyincorporated as if fully set forth herein. In some cases it wasnecessary to further purify oligodeoxynucleotides by C18-Sep-Pakchromatography (Waters Associates, Millipore, Inc.), as describedpreviously in Kern et al., J. Clin. Invest., 81:237-244 (1988), toeliminate nonspecific cytotoxic activity. Oligodeoxynucleotides elutedin 30% acetonitrile were evaporated to dryness, resuspended at 1-2 mM insterile Dulbecco's phosphate-buffered saline or Hanks' buffered saltsolution (both from Gibco), and stored at -80° C. in small aliquots.

Cells and Cell Cultures

Human leukemic cells lines used for these studies were RS11846follicular lymphoma cells, 697 pre-B cell acute lymphocytic leukemiccells, and JURKAT T cell acute lymphocytic leukemic cells as describedin Tsujimoto et al., Proc. Natl. Acad. Sci. USA, 83:5214-5218 (1986) andWeiss et al., J. Immunol., 138:2169-2174 (1987). Human peripheral bloodlymphocytes (PBL) were isolated from fresh whole blood as described inReed et al., J. Immunol., 134:314-319 (1985). All lymphcid cells werecultured at 5×10⁵ cells/ml in RPMI medium supplemented with 1 mMglutamine, antibiotics, and either 5-10% (v:v) fetal bovine serum (FBS),5-10% (v:v) calf serum (CS) (both from Hyclone Laboratories), or 1%(v:v) HLI concentrated supplement (Ventrex Laboratories) for serum-freecultures. Murine fibroblast cell lines were added at 10³ cells/cm² inDMEM medium containing glutamine, antibiotics and 5-10% (v:v) FCS.Fibroblast cell lines were NIH 3T3 cells, 3T3-B-alpha-S cells, and3T3-B-alpha-AS cells. These latter two cell lines are NIH 3T3 cells thatexpress high levels of a human bcl-2-alpha cDNA in either the sense orantisense orientation, respectively, by virtue of stable transfectionwith expression vectors constructs.

Measurement of Cellular Proliferation

Proliferation of cell lines cultured in the presence or absence ofoligodeoxynucleotides was measured by two methods: cell counts using ahemocytometer; and DNA synthesis by assaying ³ H!-thymidineincorporation essentially as described in Reed et al., J. Immunol.,134:314-319 (1985). Briefly, cells were cultured in 96-wellflat-bottomed microtiter plates (Falcon) at 0.2 ml/well. At appropriatetimes, cells were resuspended, 25 μl removed from cultures for cellcounting, and this volume replaced with 25 μl of 20 uCi/ml ³H!-thymidine (specific activity 6.7 Ci/mmole) (New England Nuclear).Microtiter cultures were then returned to 37° C. and 95% air: 5% CO₂atmosphere for 8 hours before lysing cells on glass filters anddetermining relative levels of ³ H!-thymidine incorporation into DNA byscintillation counting. Cell counts were performed in the presence oftrypan blue dye to determine the concentration of viable cells induplicate microcultures.

RNA Blot Analysis

Total cellular RNA was isolated by a guanidine-isothiocyanate/phenolprocedure as described in Chomczynski et al., Analyt. Biochem.,162:156-159 (1987). The polyadenylated fraction was purified byoligodeoxythymidine-cellulose chromatography as described in Aviv etal., Proc. Natl. Acad. Sci. USA, 69:1408-1412 (1972). Approximately 5 μgaliquots of mRNA were size-fractionated in 0.8% agarose/6% formaldehydegels and transferred to nylon membranes. Blots were prehybridized,hybridized, and washed exactly as described in Reed et al., Mol. CellBiol., 5:3361-3366 (1985), using either a ³² P-cDNA for human bcl-2, asdescribed in Tsujimoto et al., Proc. Natl. Acad. Sci. USA, 83:5214-5218(1986), or a murine bcl-2 probe, pMBCL5.4 as described in Negrini etal., Cell, 49:455-463 (1987). Blots were exposed to Kodak XAR film withintensifying screens at -70° C. for 1-10 days. Eluting ³² P-bcl-2 probesfrom membranes and rehybridizing with a ³² p probe for mousebeta-2-microglobulin verified nearly equivalent amounts of mRNA for allsamples on blots.

RESULTS Synthetic Oligodeoxynucleotides

Table 1 shows the oligodeoxynucleotides synthesized and their relationto the sense-strand of the human bcl-2 gene. Portions of the sequence ofthe coding strand of the human bcl-2 gene are shown, including thetranslation initiation site (top), splice donor site (middle), andsplice acceptor region (bottom).

The sequences of the oligodeoxynucleotides synthesized for theseinvestigations are presented, and their relation to human bcl-2mRNA isindicated. The TI-AS oligodeoxynucleotide is antisense at thetranslation initiation site and TI-S is its complementary sense version.SD-AS and SD-S are oligodeoxynucleotides having antisense and senseorientations, respectively, relative to the splice donor region.

The oligodeoxynucleotide TI-AS straddles the predictedtranslation-initiation site of bcl-2 mRNAs and is complementary(antisense) to this region. As a control, the sense version of this 20bp oligodeoxynucleotide, TI-S, was also synthesized.

In an effort to specifically block splicing of bcl-2 mRNAs, a 20 bpantisense oligodeoxynucleotide, SD-AS, was synthesized that overlaps thesplice donor site in bcl-2 primary transcripts. In addition, acomplementary sense oligodeoxynucleotide, SD-S, was prepared as depictedin Table 1. The human bcl-2 gene gives rise to several transcriptsthrough alternative splice site selections, see Tsujimoto et al., Proc.Natl. Acad. Sci. USA, 83:5214-5218 (1986). The preponderance of thesetranscripts depend upon splicing and encode a 26 kDa protein,bcl-2-alpha. One minor transcript, however, does not undergo a spliceand consequently encodes a 22 kDa protein bcl-2-beta. The SD-ASoligodeoxynucleotide can thus potentially block maturation of most butnot all bcl-2 transcripts.

Treatment of Serum for In Vitro Investigations of Antisense NormalOligodeoxynucleotides

Because normal oligodeoxynucleotides are sensitive to degradation bynucleases present in serum, the efficacy of the TI-ASoligodeoxynucleotide in fetal bovine serum (FBS) heated for 30 minutesat 56° C. (the usual procedure for inactivating serum complement) wascontrasted with the efficacy of TI-AS in FBS heated for 1 hour at 68°C., a temperature sufficient for irreversible inactivation of manynucleases. The RS11846 follicular lymphoma cell line was used. RS11846cells contain a t (14; 18) chromosomal translocation that deregulatesbcl-2 expression, resulting in the accumulation of high levels of bcl-2mRNAs, Tsujimoto et al., Proc. Natl. Acad. Sci. USA, 83:5214-5218(1986).

RS11846 follicular lymphoma cells were cultured in medium containing 5%(vol:vol) fetal bovine serum (FBS) that had been heated at 56° C. for0.5 hours or at 68° C. for 1 hour. TI-AS normal oligodeoxynucleotide wasadded at the initiation of culture, and the density of viable cellsdetermined two days later.

The TI-AS normal oligodeoxynucleotide was more effective in 68°C.-treated serum at suppressing the growth in culture of these lymphomacells. In all subsequent experiments, sera heated at 68° C. for 1 hourprior to use were used in cultures. This treatment did not impair thegrowth-supporting capacity of the sera.

Specific Inhibition of Lymphoid Cell Growth by Antisense NormalOligodeoxynucleotides

Antisense normal oligodeoxynucleotides directed against the translationinitiation site (TI-AS) and the splice donor site (SD-AS) of bcl-2transcripts were tested for their ability to suppress the proliferationof normal and neoplastic lymphoid cells.

RS11846 follicular lymphoma cells, JURKAT T cell leukemia cells, andfreshly isolated peripheral blood lymphocytes were cultured in mediumcontaining 10% (vol:vol) FBS that had been heated at 68° C. for onehour. Various concentrations of normal oligodeoxynucleotides were addedat the initiation of culture, including: TI-AS, TI-S, SD-AS, and SD-S.Relative DNA synthesis was measured in cultures after 2-3 days by ³H!-thymidine incorporation. Data were calculated as a percentage ofcontrol cultures containing volumes of PBS or HBSS equivalent tooligodeoxynucleotide-treated cultures, and represent the mean (±standard deviation) of duplicate cultures.

Similar data were obtained by measuring cell counts, excluding coldthymidine inhibition as an explanation for the suppression of DNAsynthesis observed in cultures treated with antisenseoligodeoxynucleotides.

As shown in FIG. 1, both the TI-AS and SD-AS oligodeoxynucleotidesinhibited the growth of RS11846 cells in a concentration-dependentmanner. The SD-AS oligonucleotide was less effective in inhibiting cellgrowth than the TI-AS oligodeoxynucleotide. In contrast to theseantisense oligodeoxynucleotides, sense oligodeoxynucleotides (TI-S andSD-S) were not inhibitory even at concentrations of up to 250 μg/ml.Moreover, non-sense oligodeoxynucleotides (i.e., those having the samebase composition as the antisense oligodeoxynucleotides but withscrambled sequences) also failed to suppress the proliferation ofRS11846 cells. The data thus indicate that antisenseoligodeoxynucleotides can specifically block the proliferation of thesetumor cells. Several other leukemic cell lines that express the bcl-2gene were also tested for inhibition of their proliferation by TI-AS andSD-AS oligonucleotides. As with the JURKAT T cell acute lymphocyticleukemic cells, in every case a specific and concentration-dependentdecrease in the growth of these human leukemic cells in culturescontaining antisense oligodeoxynucleotides was observed.

It has been demonstrated that bcl-2 expression is transiently induced innormal human peripheral blood lymphocytes (PBL) when these cells arestimulated to proliferate, suggesting that this gene may play a role inthe regulation of normal lymphocyte growth, Reed et al., Science236:1295-1297 (1987). The capacity of antisense oligodeoxynucleotides toimpair the growth of PBL cultured with a monoclonal antibody, OKT3 (Vanden Elsen et al., Nature 312:413-418 (1984)), that stimulates theirproliferation was therefore tested. PBL were stimulated with 50 ng/ml ofpurified OKT3 monoclonal antibody. As shown in FIG. 1, the TI-ASoligodeoxynucleotide specifically suppressed the proliferation of PBL ina concentration-dependent manner. These antisense normaloligodeoxynucleotides thus suppressed the growth in culture of leukemiccells that constitutively express the bcl-2 gene and of normallymphocytes wherein bcl-2 expression is inducible.

Time-course of Inhibition by Antisense Normal Oligodeoxynucleotides

The kinetics of inhibition by antisense oligodeoxynucleotides wasexamined in cultures of RS11846 follicular lymphoma cells and of 697pre-B cell acute lymphocytic leukemia cells. Both of these neoplastic Bcell lines transcribe and accumulate bcl-2 mRNAs at high levels,Tsujimoto et al., Proc. Natl. Acad. Sci. USA, 83:5214-5218 (1986).

RS11846 follicular lymphoma and 697 pre-B cell leukemia cells werecultured in medium containing 10% (vol:vol) 68° C.-treated FBS andnormal oligodeoxynucleotides. Cells were cultured with 50 μg/ml TI-AS,100 μg/ml SD-AS, 50 μg/ml TI-S (RS11846 cells) or 100 μg/ml SD-S (697cells), or PBS as a control. DNA synthesis (kcpm/10⁶ viable cells) andcell densities (10⁶ viable cells/ml) were measured at various timesafter initiation of cultures.

Antisense normal oligodeoxynucleotides markedly inhibited DNA synthesismeasured in cultures of these cells within 24 hours. Diminished celldensities were readily apparent in these cultures within 2 days.Antisense normal oligodeoxynucleotides thus rapidly inhibited the invitro growth of leukemic cells. The action of antisenseoligodeoxynucleotides was specific, since sense oligodeoxynucleotidesdid not impair proliferation in these cultures. Though cell viabilitiesoften declined during the later days of culture no increase in celldeath was seen during the first 1-2 days of culture with antisenseoligodeoxynucleotides, suggesting a non-cytotoxic mechanism.

Comparison of Different Serum Preparations

Inhibition of proliferation of leukemic cells with antisenseoligodeoxynucleotides can vary greatly depending on the lot of serumused in cultures.

To determine the effects of serum of inhibition of proliferation,relative levels of DNA synthesis were measured in cultures of 697 pre-Bcell leukemia cells 2 days after addition of 200 μM TI-AS normaloligodeoxynucleotide. Cells were cultured in medium supplemented with 1%(vol:vol) HL1-concentrate (serum-free condition), 5% (vol:vol) of twodifferent lots of calf serum (CS1 and CS2), or 5% (vol:vol) of twodifferent lots of fetal bovine serum (FBS1 and FBS2). All sera wereheated at 68° C. for 1 hour prior to use in cultures.

The normal TI-AS oligodeoxynucleotide markedly inhibited DNA synthesis(92%) and cellular proliferation in serum-free cultures (HL1) of 697cells. This antisense oligodeoxynucleotide was equally effective (94%)in cultures containing 5% (v:v) of one of the lots of fetal bovine serum(FBS2). In contrast, inhibition was significantly reduced in culturescontaining other serum preparations (CS1, CS2, FBS1). It has beengenerally observed that antisense normal oligodeoxynucleotides are lesseffective in cultures supplemented with calf serum (CS) than in thosecontaining fetal bovine serum (FBS).

Concentration Dependence of Inhibition by Antisense NormalOligodeoxynucleotides in Serum-free Cultures

697 pre-B cell leukemia cells were cultured in medium with either 1%(vol:vol) HL1-concentrate (serum-free conditions or 5% (vol:vol) 68°C.-treated FBS2). Relative levels of DNA synthesis and cellulardensities measured after 2 days in cultures containing variousconcentrations of normal TI-AS oligodeoxynucleotide.

The TI-AS oligodeoxynucleotide was inhibitory at lower concentrationswhen used in serum-free cultures. At 100 μM, for instance, no inhibitionof cellular proliferation was seen in FBS2-containing cultures, whereascell counts were reduced by approximately 75% in serum-free cultures. Athigher concentrations of antisense oligodeoxynucleotide (200-250 μM),however, inhibition of 697 cellular proliferation was comparable in bothtypes of cultures. The increased efficacy of normaloligodeoxynucleotides in serum-free cultures was specific, since thesense oligonucleotide (TI-S) was not inhibitory at the sameconcentrations.

Antisense Phosphorothloate Oligodeoxynucleotides: Time-Course ofInhibition

To contrast the efficacy of phosphorothioate oligodeoxynucleotides withthat of normal oligonucleotides with regard to inhibition of humanleukemic cell growth, phosphorothioate oligodeoxynucleotides werecultured with 697 pre-B cell leukemia cells and the effects oninhibition were measured. 697 pre-B cell leukemia cells were cultured inserum-free medium for various times before measuring DNA synthesis(kcpm) and cell densities (10⁶ cells/ml). Cells were seeded at aninitial density of either 0.2×10⁶ cells/ml or 0.5×10⁶ cells/ml. Cultureconditions were 25 μM TI-AS phosphorothioate, 25 μM TI-Sphosphorothioate, and control cultures treated with HBSS.

To avoid experimental variation due to differences among lots of sera,697 leukemic cells were cultured in serum-free conditions. When culturedat an initial seeding density of 0.5×10⁶ cells/ml, 697 cells achievedmaximal DNA synthesis and cellular densities at 4-5 days. Addition of 25μM sense phosphorothioate oligodeoxynucleotide (TI-S) at the initiationof these cultures had little effect on 697 cell growth. In replicatecultures containing 25 μM antisense phosphorothioate (TI-AS), however,some diminution in DNA synthesis was evident within 2 days and wasmaximal at 4-5 days. Maximal inhibition of 697 cell growth, asdetermined by cell counts, was seen at 6 days after initiation ofcultures.

When 697 cells were initially seeded at 0.2×10⁶ cells/ml, the antisensephosphorothioate oligodeoxynucleotide, TI-AS, resulted in only slightinhibition at 2 days, attaining maximal suppression of DNA synthesis inthese cultures at day 7. As with normal oligodeoxynucleotides, thisinhibition by phosphorothioate oligodeoxynucleotides appeared to bemediated through non-cytotoxic mechanisms, since cellular viabilitiesdid not decline until late in the course of culture. Compared withnormal antisense oligodeoxynucleotides, therefore, phosphorothioateoligodeoxynucleotides had a slower onset of action.

Concentration Dependence of Inhibition by Antisense bcl-2Phosphorothioate Oligodeoxynucleotides

The concentration dependence of inhibition by phosphorothioate andnormal TI-AS oligodeoxynucleotides in cultures of 697 cells inserum-free medium was compared as follows.

697 cells were cultured in serum-free medium for either 3 days (normaloligodeoxynucleotides) or 4 days (phosphorothioateoligodeoxynucleotides) prior to measuring cell densities and levels ofDNA synthesis. Oligodeoxynucleotide additions to cultures included TI-ASphosphorothioate, TI-S phosphorothioate, TI-AS normal, and TI-S normal.

As shown in FIG. 2, TI-AS phosphorothioate oligodeoxynucleotidesmarkedly inhibited the proliferation of 697 cells at 25-50 μM. Incontrast, normal TI-AS oligodeoxynucleotides required concentrations5-10-fold higher (approximately 250 μM) to cause a comparablesuppression of 697 cellular proliferation. Suppression by the antisensephosphorothioate oligodeoxynucleotide TI-AS was specific over thisconcentration range, since its complementary sense oligodeoxynucleotide(TI-S) produced little inhibition of 697 cell growth in replicatecultures (see FIG. 2).

Influence of Serum Preparation on Inhibition by AntisensePhosphorothioate Oligodeoxynucleotides

To further define the effects of serum preparation on the inhibitoryactivity of phosphorothioate oligodeoxynucleotides, FBS that had beenheated to 56° C. for 30 minutes, 68° C. for 1 hour, or not heated priorto addition to cultures was added to cultures of RS11846 lymphoma cells.

RS11846 cells were cultured in medium containing 1% (vol:vol)HL1-concentrate or 5% (vol:vol) FBS that had been heated at 56° C. for0.5 hour, 68° C. for 1 hour, or that had not been heated. Cell countswere calculated as a percentage relative to control cultures treatedwith equivalent concentrations of TI-S phosphorothioateoligodeoxynucleotide, and represent the mean percentage (standarddeviation was less than 10% for all values) for duplicate culturescounted on days 4 and 5.

The TI-AS phosphorothioate oligodeoxynucleotide completely inhibited thegrowth of RS11846 cells at 25 μM, with an estimated half-maximalinhibitory concentration of approximately 11 μM. In contrast, thisphosphorothioate oligodeoxynucleotide was considerably less effective incultures containing 5% (v:v) FBS. Furthermore, heating FBS prior toadding it to cultures did not significantly improve the ability of theTI-AS phosphorothioate oligodeoxynucleotide to suppress the growth ofRS11846 lymphoma cells. At an oligodeoxynucleotide concentration of 50μM, inhibition of proliferation of RS11846 cells never exceeded 48% inserum-containing cultures, regardless of the heating procedure used.

Influence of Dialysis of Serum on Inhibition by Normal andPhosphorothioate Antisense Oligodeoxynucleotides

To further characterize the nature of the interfering substances inserum, experiments were performed wherein 68° C.-heated serum wasextensively dialyzed (molecular weight cutoff=3500) prior to being addedto cultures of 697 leukemic cells. Experiments were conducted with 12.5μM TI-AS phosphorothioate oligodeoxynucleotide and 200 μM of the normaloxygen-based TI-AS oligodeoxynucleotide.

697 cells were cultured in medium containing 1% (vol:vol)HL1-concentrate (A) or 5% (vol:vol) of three different lots of 68°C.-treated FBS (B,C,D). Each serum preparation was contrasted before(ND) and after (D) extensive dialysis. TI-AS (+) and TI-S (-)oligodeoxynucleotides were added to replicate cultures at 200 μM fornormal oxygen-based oligodeoxynucleotides (OXY) and at 12.5 μM forphosphorothioate oligodeoxynucleotides (PT). Relative levels of DNAsynthesis (kcpm) were measured after 2 or 4 days of culture for normaland phosphorothioate oligodeoxy-nucleotides, respectively.

For the three different lots of FBS tested, two exhibited little changeafter dialysis in cultures containing either normal or phosphorothioateoligodeoxynucleotides. One lot of FBS, however, appeared to interfereless with the inhibitory activities of these antisenseoligodeoxynucleotides after dialysis.

Experiments with Stably Transfected NIH 3T3 Cells

Though the antisense oligodeoxynucleotides described herein weredesigned to block bcl-2 mRNA translation (TI-AS) and splicing (SD-AS),the molecular mechanisms of their actions are not yet known. Todetermine the effect of formation of oligodeoxynucleotide-RNA hybridswithin cells upon inhibition of cellular growth, irrespective of thenucleotide sequence, cells transformed to express human bcl-2 cDNAtranscripts were cultured with normal oligodeoxynucleotides.

200 μM of normal TI-AS and TI-S oligodeoxynucleotide were added tocultures of typical NIH 3T3 cells and to cultures of these cells thathad been stably transfected with expression constructs that produce highlevels of human bcl-2 cDNA transcripts for either the usual sense(3T3-alpha-S cells) or the antisense (3T3-alpha-AS cells) strand.

For RNA blot analyses, polyadenylated mRNA was purified from normal NIH3T3 cells and from cells stably transfected with expression constructsthat produce either sense (3T3-alpha-S) or antisense (3T3-alpha-AS)recombinant bcl-2-alpha mRNAs, according to the method of 13.Approximately 5 μg of mRNA was subjected to RNA blot analysis,essentially as described in (16), using either ³² P-labeledhybridization probes derived from human or murine bcl-2 sequences.

An autoradiogram resulting from a one-day exposure of a blot containingRNAs from normal 3T3 cells, 3T3-alpha-AS cells, and 3T3-alpha-S cellsshowed high relative levels of recombinant 2.4 and 1.4 kbp bcl-2transcripts produced from the bcl-2 expression constructs that weretransfected into 3T3-alpha-AS and 3T3-alpha-S cells.

A 10-day exposure of a blot containing RNA from normal 3T3 cells thatwere either proliferating or quiescent at the time of harvesting RNAshowed low but detectable levels of normal 7.5 and 2.4 kbp murine bcl-2transcripts present in proliferating 3T3 cells.

TI-AS oligodeoxynucleotide specifically suppressed DNA synthesis andcellular replication in cultures of normal NIH 3T3 cells, consistentwith findings by others that fibroblasts do contain bcl-2 transcripts,albeit at low levels. The TI-AS oligodeoxynucleotide disclosed herein iscomplementary to the mouse bcl-2 sequence in 18 of its 20 bases (17),accounting for its ability to suppress the growth of murine NIH 3T3cells.

NIH 3T3 cells, 3T3-alpha-AS cells, and 3T3-alpha-S cells were culturedin medium containing 5% (vol:vol) 68° C.-treated serum and either HBSS,200 μM TI-S normal oligodeoxynucleotide, or 200 μM TI-AS normaloligodeoxynucleotide. Relative levels of DNA synthesis (kcpm) weremeasured in cultures after 3 days and reflect a 16 hour incubation with0.5 μCi/well of ³ H!-thymidine. Cell densities, estimated by phasemicroscopy, were consistent with the measured DNA synthesis in cultures.The percentage of inhibition of DNA synthesis in cultures containingTI-AS oligodeoxynucleotides was calculated relative to control culturescontaining HBSS.

As with normal NIH 3T3 cells, culturing 3T3-alpha-S cells (producinghuman bcl-2-alpha sense transcripts) with TI-AS and TI-Soligodeoxynucleotides demonstrated specific suppression, since the senseoligodeoxynucleotide TI-S was not inhibitory. The level of inhibition ofcellular proliferation by the antisense oligodeoxynucleotide, however,was not as great in 3T3-alpha-S cells, as might be expected, since thesecells contain more bcl-2 mRNA.

Adding TI-S oligodeoxynucleotide to cultures of 3T3-alpha-AS cells(produce antisense bcl-2 transcripts) ruled out inhibition of cellulargrowth through a nonspecific mechanism involvingoligodeoxynucleotide-RNA hybrid formation. The TI-S oligodeoxynucleotidecaused little suppression of 3T3-alpha-AS cell proliferation, whereasthe TI-AS oligodeoxynucleotide was markedly inhibitory in these cells.Similar data were obtained with TI-AS and TI-S phosphorothioateoligodeoxynucleotides.

Measurements of DNA Fragmentation As An Indicator Of bcl-2 AntisenseOligodeoxynucleotide-Mediated Programmed Cell Death In Human LymphomaCells

Oligonucleotides having the sequences shown in Table 2 were tested forthe ability to induce programmed cell death (DNA fragmentation) in thehuman t (14:18)-containing human lymphoma cell line RS11846. Theoligonucleotides were all phosphodiesters, and were targeted against thetranslation initiation site or the 5'-cap region of bcl-2 mRNAs. Controloligonucleotides included a bcl-2 sense version (TI-S) of TI-AS (havingSEQ ID NO: 7) and a scrambled version of TI-AS that has the same basecomposition, but with jumbled nucleotide order.

                  TABLE 2                                                         ______________________________________                                        SEQUENCE           SEQ ID NO:                                                 ______________________________________                                        CGCGTGCGAC CCTCTTG  8                                                         TACCGCGTGC GACCCTC  9                                                         TCCTACCGCG TGCGACC 10                                                         CCTTCCTACC GCGTGCG 11                                                         GACCCTTCCT ACCGCGT 12                                                         GGAGACCCTT CCTACCG 13                                                         GCGGCGGCAG CGCGG   14                                                         CGGCGGGGCG ACGGA   15                                                         CGGGAGCGCG GCGGGC  16                                                         ______________________________________                                    

RS11846 cells were adapted to grow in HL1 media with 1% FCS and theirDNA metabolically labeled by addition of ¹²⁵ I-deoxyuridine to culturesfor three hours. Labeled cells were then washed thoroughly and culturedfor two days in the presence of various oligonucleotides at 50 μM. Cellswere then recovered from 200 μL cultures by centrifugation, and lysed ina hypotonic buffer containing 10 mM EDTA and 1% Triton X100. Aftercentrifugation at 16,000 xg to pellet unfragmented genomic DNA, thesupernatant fraction containing fragmented DNA was extracted withphenol/chloroform and ethanol precipitated. This DNA was then subjectedto gel electrophoresis in 1.5% agarose gel and transferred to nylonmembranes for autoradiography.

The results of two experiments are shown in FIGS. 3 and 4. The six bcl-2antisense oligonucleotides targeted in the vicinity of the ATG site oftranslation initiation in bcl-2mRNAs were tested. "C-Oligo-2" refers toan oligonucleotide with 4 purposeful mismatches. "U" indicates untreatedcontrol cells. FIG. 4 shows the results for the oligonucleotides shownin FIG. 3. "Sc20" refers to a 20 mer with the same base composition asTI-AS, but with scrambled sequence. FIG. 4(b) shows the results forthree oligonucleotides targeted against the 5'-cap of bcl-2 mRNAs. Thenumbers refer to the distance of these oligomers from theATG-translation initiation site.

The presence of a ladder of DNA fragments (unit size of approximately200 bp) is indicative of programmed cell death. At 50 μM, TI-AS causedlittle DNA fragmentation, whereas the oligonucleotides having SEQ ID NO:9 and SEQ ID NO: 10, and one of the 5'-cap oligonucleotides (SEQ ID NO:14) led to pronounced DNA fragmentation.

Concentration-Dependence of Inhibition by Antisense PhosphodiesterOligodeoxynucleotides in Serum-Free Cultures

697 pre-B cell leukemia cells were cultured in medium with either 1%(vol:vol) HL-1 concentrate (serum-free conditions o! or 5% (vol:vol) 68°C.-treated serum (FBS2) !, see FIG. 5. Shown are cellular densitiesmeasured after 2 days in cultures containing various concentrations ofphosphodiester TI-AS oligodeoxynucleotide. Data are shown as percentagesrelative to control cultures treated with a sense oligonucleotide, andreflect the mean ± standard deviation for duplicate samples.

Immunofluorescence Analysis of bcl-2 Protein Levels inOligodeoxynucleotide-Treated 697 Cells

For studies with oligodeoxynucleotides, 0.25×10⁴ (for phosphorothioate)or 0.5×10⁶ (for normal oligodeoxynucleotides), 697 cells were culturedin 1 ml of HL-1 serum-free medium in 24 well culture dishes (Linbro.Flow Lab, Inc.). After 2 days (for normal) or 4 days (forphosphorothioates), cells were recovered from cultures, washed once inPBS-A PBS, pH 7.4 (Gibco)-0.1% bovine serum albumin-0.1% sodium azide!,and fixed for 5-10 minutes on ice in 1% paraformaldehyde/PBS solution.The cells were then washed once in PBS and incubated in 1 ml of absolutemethanol at -20° C. for 10 minutes. After washing once in PBS-A, cellswere then resuspended in PBS containing 0.05% Triton-X100 for 3 minuteson ice, washed in PBS-A and preblocked for 30 minutes at 4° C. in PBSwith 10% (v/v) heat-inactivated goat serum.

For addition of the first antibody, preblocked cells were resuspended in100 μl of PBS-G (PBS-1% goat serum-0.1% sodium azide) prior toaliquoting 50 μl into separate tubes that contained 1 μl of either BCL2antibody (Halder et al., Nature (London), 342:195-197 (1989)) oraffinity-purified normal rabbit control IgG (Cappel 6012-0080) andincubated for 1 hour on ice. The BCL2 antibody used for these studieswas prepared in rabbits using a synthetic peptide corresponding to aminoacids (98-114) of the BCL2 protein and was affinity-purified byprotein-A-Sepharose chromatography and used at approximately 1 mg/ml.Cells were then washed in PBS-A and incubated in 0.5-1.0 ml PBS-A for15-20 minutes on ice to allow diffusion of nonspecific cell-associatedantibody prior to resuspending cells in 100 μl of PBS-G containing 5 μgof biotinylated goat anti-rabbit IgG (BA1000; Vector Labs) for 30minutes. After washing once and incubating for 15 minutes in PBS-A,cells were finally resuspended in 100 μl of PBS-A containing 2 μg ofFITC-conjugated avidin (Vector Labs A2011) for 20 minutes and washedthree times in PBS-A prior to analysis with an Ortho cytofluorograph50-H connected to an Ortho 2150 data-handling system. The specificity ofthis method for detecting BCL2 protein was confirmed byimmunofluorescence microscopy (showing cytosolic staining), peptidecompetition, and studies of cell lines that expressed various levels ofBCL2mRNA and proteins through gene transfer manipulations.

For measurements of surface HLA-DR antigen expression, an indirectimmunofluorescence assay method was used (Reed et al., J. Immunolo,134:1631-1639 (1985)) involving incubation of viable cells with a murineanti-HLA-DR monoclonal antibody (IgG2a)(Becton-Dickinson 7360) or anegative control antibody, R3-367 (IgG2a), followed by FITC-conjugatedgoat anti-mouse IgG (Cappel 1711-0081). Cells were fixed in 1%paraformaldehyde/PBS prior to FACS analysis.

697 cells were cultured for 2 days (PO) or 4 days (PS) with variousoligonucleotides. In FIG. 6, the black columns show the results with asense oligonucleotide, and the hatched columns with an antisenseoligonucleotide TI-AS. Cells were labeled with anti-bcl-2 antiserum andanalyzed by FACS. Data are expressed as percentages relative to the meanfluorescence obtained with untreated 697 cells.

FIG. 7 shows typical FACS results obtained for 697 cells before andafter treatment with 100 μM PO bcl-2 antisense oligonucleotides. A:untreated 697 cells labeled with either anti-bcl-2 antiserum (hatchedarea) or normal rabbit serum control (white area); B: untreated 697cells labeled with either anti-HLA-DR antibody (hatched area) or anegative control antibody (white area); C: 697 cells cultured for 2 dayswith either normal bcl-2 TI-AS (white area) or TI-AS (hatched area)oligodeoxynucleotides and labeled with anti-bcl-2 antibody; D: 697 cellscultured with TI-AS and TI-S oligodeoxynucleotides (as in C), butlabeled with anti-HLA-DR antibody.

As shown in FIGS. 6 (a) and (b), PO and PS bcl-2 antisenseoligonucleotides produced specific concentration-dependent reductions inthe levels of bcl-2 proteins, without altering the levels of expressionof HLA-DR (FIG. 7) and other control antigene. At 150 μM, for example,PO antisense oligodeoxynucleotide caused an approximately 75-95%reduction in bcl-2 fluorescence, whereas the control senseoligodeoxynucleotide diminished bcl-2 protein levels by only 10-20%(FIG. 6(a)). Similarly, cultured 697 cells for 4 days with the PSantisense oligodeoxynucleotide ar 25 μM resulted in approximately 70%reduction in bcl-2 fluorescence. In comparison, the sense PSoligodeoxynucleotide TI-AS inhibited bcl-2 protein levels by onlyapproximately 15%, as measured by this assay (FIG. 6(b)).

SIGNIFICANCE

In phosphorothioate oligodeoxynucleotides one of the non-bridging oxygenatoms in each internucleotide phosphate linkage is replaced by a sulfuratom. This modification renders phosphorothioate oligodeoxynucleotidesextremely resistant to cleavage by nucleases, Stein et al., Nucl. AcidsRes., 16:3209-3221 (1988). Despite the substitution of a sulfur atom foran oxygen, phosphorothioate oligodeoxynucleotides retain good solubilityin aqueous solutions; hybridize well, though with some decrease in themelting temperature of RNA-oligodeoxynucleotides duplexes; and aresynthesized conveniently by the widely employed method of automatedoligodeoxynucleotides synthesis with phosphoroamidites.

Antisense bcl-2 phosphorothioate oligodeoxynucleotides have been foundto be more potent inhibitors of leukemic cell grown than their normaloxygen-based counterparts. When tested under serum-free conditions,these oligodeoxynucleotides reduced cellular proliferation by half atconcentrations of approximately 15-25 μM, whereas the normaloligodeoxynucleotide achieved 50% inhibition at 125-250 μM. This findingmay be explained by the reduced sensitivity of phosphorothioateoligodeoxynucleotides to cellular nucleases, or may be attributable toother mechanisms. For example, mRNAs hybridized with phosphorothioateoligodeoxynucleotides may experience enhanced degradation through amechanism involving an RNAse H-like activity.

Despite their increased inhibitory activity, phosphorothioate antisenseoligodeoxynucleotides retained sequence-specificity. At theconcentrations tested (less than 25 μM), sense versions of theseoligodeoxynucleotides had little effect on leukemic cell growth. Bothnormal and phosphorothioate antisense oligodeoxynucleotides appeared toinitially suppress the proliferation of leukemic cells throughnon-cytotoxic mechanisms. During the first few days of culture, cellularreplication was inhibited without a concomitant rise in cell death.Later in these cultures (days 4-5 for normal oligodeoxynucleotides, days6-8 for phosphorothioates), however, cellular viabilities declined.

Comparing the kinetics of inhibition by normal and phosphorothioateoligodeoxynucleotides revealed that the latter compounds have a sloweronset of action. Maximal inhibition of leukemic cell proliferation bynormal antisense oligodeoxynucleotides occurred two days afterinitiation of cultures, whereas phosphorothioate oligodeoxynucleotidesrequired 4 to 7 days to achieve maximal inhibition.

The usefulness of antisense oligodeoxynucleotides in inhibiting humanlymphoma/leukemia cells that express the bcl-2 gene has been shown bythe examples herein. Antisense oligodeoxynucleotides complementary to atleast an effective portion of the mRNA of the human bcl-2 gene has beenfound to inhibit proliferation of RS11846 human follicular lymphomacells (t (14;18) chromosomal translocation and high bcl-2 expression),697 human pre B cell leukemia cells (high bcl-2 expression), JURKAThuman acute lymphocytic leukemia cells (medium bcl-2 expression), normalhuman lymphocytes (medium bcl-2 expression) and murine fibroblasts (lowbcl-2 expression). Although bcl-2 antisense reagents can suppress thegrowth of many types of cells, the t (14:18)-containing lymphoma andleukemia cells seem to be the most sensitive, allowing for specificinhibition of malignant cells.

A variety of chemically modified forms of DNA can be employed in theinstant invention. For example, phosphorothioates and methylphosphonateshave been found to be useful in the present invention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 17                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: Synthetic DNA                                                (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CAGCGTGCGCCATCCTTCCC20                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CTTTTCCTCTGGGAAGGATGGCGCACGCTGGGAGA35                                         (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: Synthetic DNA                                                (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GATGCACCTACCCAGCCTCC20                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ACGGGGTACGGAGGCTGGGTAGGTGCATCTGGT33                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: Synthetic DNA                                                (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ACAAAGGCATCCTGCAGTTG20                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CCCCCAACTGCAGGATGCCTTTGTGGAACTGTACGG36                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: Synthetic DNA                                                (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GGGAAGGATGGCGCACGCTG20                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: Synthetic DNA                                                (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GTTCTCCCAGCGTGCGC17                                                           (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: Synthetic DNA                                                (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CTCCCAGCGTGCGCCAT17                                                           (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: Synthetic DNA                                                (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CCAGCGTGCGCCATCCT17                                                           (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: Synthetic DNA                                                (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GCGTGCGCCATCCTTCC17                                                           (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: Synthetic DNA                                                (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      TGCGCCATCCTTCCCAG17                                                           (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: Synthetic DNA                                                (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GCCATCCTTCCCAGAGG17                                                           (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: Synthetic DNA                                                (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GGCGCGACGGCGGCG15                                                             (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: Synthetic DNA                                                (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      AGGCAGCGGGGCGGC15                                                             (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: Synthetic DNA                                                (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      CGGGCGGCGCGAGGGC16                                                            (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: Synthetic DNA                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      TCTCCCAGCGTGCGCCAT18                                                          __________________________________________________________________________

What is claimed is:
 1. An antisense oligonucleotide complementary toBCL-2 mRNA consisting of from 10 to 35 bases and complementary to atleast 10 consecutive bases of SEQ ID NO:
 2. 2. The antisenseoligonucleotide of claim 1, wherein said oligonucleotide has 10 to 20bases.
 3. The antisense oligonucleotide of claim 1, wherein saidoligonucleotide has 20 to 35 bases.
 4. The antisense oligonucleotide ofclaim 1, which consists of from 10 to 18 bases and which iscomplementary to SEQ ID NO:
 2. 5. The antisense oligonucleotide of claim1, which is a phosphorothioate derivative.
 6. An antisenseoligonucleotide having the nucleotide sequence of SEQ ID. NO: 1, 3, 5,9, 10 or
 14. 7. The antisense oligonucleotide of claim 6, which is aphosphorothioate derivative.
 8. The antisense oligonucleotide of claim6, whose sequence is SEQ ID NO:
 1. 9. The antisense oligonucleotide ofclaim 6, whose sequence is SEQ ID NO:
 3. 10. The antisenseoligonucleotide of claim 6, whose sequence is SEQ ID NO:
 5. 11. Theantisense oligonucleotide of claim 6, whose sequence is SEQ ID NO: 9.12. The antisense oligonucleotide of claim 6, whose sequence is SEQ IDNO:
 10. 13. The antisense oligonucleotide of claim 6, whose sequence isSEQ ID NO: 14.